Polypeptide compositions and methods

ABSTRACT

A method of preparing a bioactive polypeptide in a stable, inactivated form, the method comprising the step of treating the polypeptide with ozonated water in order to oxidize and/or stabilize the cysteine residues, and in turn, prevent the formation of disulfide bridges necessary for bioactivity. The method can involve the use of ozonated water to both oxidize the disulfide bridges in a bioactive polypeptide, and to then stabilize the resultant cysteine residues. Optionally, and preferably, the method can involve the use of ozonated water to stabilize the cysteine residues, and thereby prevent the formation of disulfide bridges, in a polypeptide produced by recombinant means in a manner that allows the polypeptide to be recovered with the disulfide bridges unformed.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of US patentapplication filed Aug. 5, 1999 and assigned Ser. No. 09/368,834, whichis a continuation of US patent application filed Aug. 7, 1997 andassigned Ser. No. 08/908,212, which is a continuation of US patentapplication filed May 10, 1996 and assigned Ser. No. 08/644,399, theentire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] In one aspect, the present invention relates to methods forpreparing bioactive polypeptides in an inactive form. In another aspect,the present invention relates to bioactive polypeptides such asneurotoxins, and to methods for the preparation of such neurotoxins. Inyet another aspect, the invention relates to the use of inactivatedneurotoxin compositions for the study and treatment of viral andneurological diseases.

BACKGROUND OF THE INVENTION

[0003] Bioactive polypeptides are typically obtained by either therecovery and purification of natural products, or by synthesis using itsgenetic counterpart. Typically, the polypeptides, whether purified fromnatural sources or synthesized using recombinant technology, areultimately provided in a form having the intended bioactivity.

[0004] Occasionally, however, it is desirable to prepare otherwisebioactive polypeptides in their inactive form, in which they can be usedfor other in vivo purposes, such as the preparation of vaccines. Inother situations, the bioactivity of the polypeptide itself may be aparticularly toxic one, so as to make the recovery of the activepolypeptide either unnecessary, or unduly difficult and dangerous.

[0005] Typically, even toxic polypeptides are first recovered in theirnative, active forms, and thereafter subjected to processes intended toeither temper the bioactivity, or render the polypeptide completelyinactive. Examples of such processes include heating the polypeptide(e.g., denaturation), oxidation (e.g., by peroxide, catalase treatment),and the like. Such processes, however, are typically non-specific innature, generally irreversible, and potentially quite damaging to thepolypeptide.

[0006] Certain proteins can be inactivated by the cleavage of disulfidelinkages, for instance using a suitable reducing agent (e.g.,2-mercaptoethanol) to provide a corresponding pair of cysteine residues.Cleavage of disulfide linkages within a protein will typically result inthe unfolding of the protein. Unless maintained in the cleaved andunfolded state (e.g., in the presence of urea), the disulfide bonds areoften able to spontaneously reform, although not always pairing the sameoriginal residues (resulting in a malfolded product).

[0007] Ribonuclease, for instance, contains four disulfide bonds thatcan each be cleaved in the manner described above. Under appropriateconditions, the molecule can spontaneously reform in a manner thatprovides 95-100% of the original activity. On the other hand, if thethree disulfide bonds of insulin are cleaved under similar conditions,the molecule will spontaneously reform to provide only 5-10% of theoriginal activity. Hence, the linear amino acid sequence of a protein isnot necessarily the sole determinant of the protein's folding patternand activity.

[0008] The recovery of neurotoxins is a prime example of thedifficulties involved in handling and using bioactive molecules. See,generally, “Cloning, Characterization, and Expression of Animal ToxinGenes for Vaccine Development”, L. A. Smith, J. Toxicol.-Toxin Reviews,9(2), 243-283 (1990). The Smith article describes, for instance, therelated properties of a number of toxins from animal origin (p. 247),and the slow progress made to date in developing such vaccines.

[0009] The venom obtained from snakes such as those of the genus Najahas been found to contain a number of different physiologically active,and potentially useful, polypeptides having enzymatic and/or toxiceffects. A number of these toxins have been purified and modified forthe purpose of determining their molecular structure and mode of action.

[0010] In order to safely use such toxins, for instance, U.S. Pat. Nos.3,888,977, 4,162,303 and 4,126,676 (each naming Sanders) disclosedetoxified venom compositions. The compositions are detoxified byoxidation using catalase or peroxide, in a manner said to retain theneurotropic activity of the modified venom compositions. The Sanderspatents discuss compositions derived from the venom of the Bungarusgenus, the Naja genus and a combination of both genuses. Included insuch patents are methods for determining the potency and atoxicity ofsuch modified neurotoxins.

[0011] Neurotoxin polypeptides in their detoxified but neurotropicallyactive form have been considered for the treatment of certain viralinfections. Detoxified polypeptides have been considered, for instance,for use in the treatment of certain disorders such as the neurologicaldisorder amyotrophic lateral sclerosis (“ALS”), a disease characterizedby slow progressive degeneration of lower motor neurons. See, forinstance, “The Use of Sanders Neurotoxoid I (Modified Snake Venom) inthe Treatment of Recurrent Herpes Simplex of the Cornea: ProgressReport”, Clark, W. B., et al., Southern Medical Journal 55(9):947-951(1962).

[0012] A variety of polypeptides, including toxins, have also beencloned and expressed by genetic engineering. See, for instance, theabove-cited Smith article, which (beginning at page 257) describes anumber of efforts directed at cloning snake venom toxin genes.

[0013] Conventional methods for preparing inactive polypeptides (e.g.,detoxified neurotoxins) continue to suffer from a number of drawbacks.Among these drawbacks are the contaminants that frequently accompany thedetoxified preparations. Another drawback relates to the fact thatneurotoxins, unlike most polypeptides of a similar size, tend to bequite soluble in solvents commonly used for protein precipitation,thereby limiting the usefulness of conventional purification techniques.Yet another drawback relates to the use of any nonspecific means forrendering a polypeptide biologically inactive, since such means canoften lead to the destruction of all properties of the molecule,including such desirable properties as immunogenicity and antiviralactivity.

[0014] What is clearly needed is a method for the preparation ofinactivated bioactive polypeptides, such as neurotoxins, in a mannerthat avoids the drawbacks associated with prior methods. To applicantsknowledge, there have been no teachings in the art of the use of geneticengineering techniques, particularly in the manner provided herein, toprepare inactive forms of bioactive molecules such as neurotoxins.

SUMMARY OF THE INVENTION

[0015] In one aspect, the present invention provides a method ofpreparing a parenteral composition comprising an inactivated bioactivepolypeptide, the method comprising the steps of:

[0016] a) identifying a polypeptide having a biological activitydependent on the presence of one or more disulfide bridges in itstertiary structure,

[0017] b) preparing a cDNA strand encoding the polypeptide,

[0018] c) expressing the cDNA under conditions in which the polypeptideis recovered in an inactive form due to the failure to form one or moredisulfide bridges, and

[0019] d) recovering the inactive polypeptide and formulating it into acomposition suitable for parenteral administration to a host.

[0020] In a further aspect, the invention provides a method ofadministering a composition comprising an inactivated bioactivepolypeptide to a host, comprising the step of providing the polypeptidein an inactive form and in a composition that facilitates itsadministration to the host. In a related aspect, the invention providesa host having administered such a composition.

[0021] In another aspect, the invention provides a compositioncomprising a bioactive polypeptide that has been rendered inactive byvirtue of the failure to form one or more of its disulfide bridges. In arelated aspect, the invention provides a composition for in vivoadministration comprising a bioactive polypeptide that has beeninactivated in the manner described herein.

[0022] The method can be used to prepare a variety of bioactivepolypeptides, including “Group I neurotoxins” (namely, toxins affectingthe presynaptic neurojunction), Group II neurotoxins (namely thoseaffecting the postsynaptic neurojunction), and Group III neurotoxins(those affecting ion channels). cDNA sequences for such polypeptides aregenerally known, or can be determined using conventional techniques.

[0023] The cDNA can be expressed using any suitable expression system,under conditions in which the product can be recovered with one or moredisulfide bridges unformed. Suitable expression systems includeheterologous host systems such as bacteria, yeast or higher eucaryoticcell lines. Examples of useful systems are described, for instance, in“Foreign Gene Expression in Yeast: a Review”, Romanos, et al., Yeast,8:423-488 (1992). See also, “Yeast Systems for the Commercial Productionof Heterologous Proteins”, Buckholz, et al., Bio/Technology 9:1067-1072(1991), the disclosures of both Romanos et al. and Buckholz et al. beingincorporated herein by reference.

[0024] These articles are generally directed at the more common goal ofaffirmatively achieving posttranslational processing and extracellularsecretion. Under such conditions, the formation of appropriate disulfidelinkages would be included as a necessary step. Given the presentdescription, however, these articles, and the techniques describedtherein, will be of considerable use to those skilled in the art inachieving the recovery of the unfolded product, e.g., by intracellularexpression in yeast.

[0025] Preferably, the cDNA is expressed using a yeast expressionsystem, such as Saccharomyces cerevisiae and Pichia pastoris. Morepreferably, the cDNA is expressed in a Pichia expression system underconditions in which the product is cytoplasmically produced, as opposedto extracellularly secreted. In an exemplary embodiment, the polypeptideis expressed using a Pichia expression system, under conditions in whichthe leader sequence of naturally-occurring cDNA is removed and replacedwith only the initiation codon.

[0026] Polypeptides of the present invention are generally stable undersuitable conditions of storage and use in which the disulfide bonds areprevented from spontaneously reforming, or are allowed to reform in amanner that precludes the undesirable activity of the polypeptide.Optionally, and preferably, once the inactive polypeptide has beenrecovered, it is treated by suitable means to ensure that the cysteineresidues do not spontaneously reform to form disulfide bridges. Anexample of a preferred treatment means is the use of ozone treatment asdescribed herein. Alternatively, as will be described in greater detailbelow, ozone treatment can itself be used to selectively break (i.e.,oxidize) the disulfide bonds of a native or recombinantly prepared toxinmolecule in order provide a stable, inactive form thereof.

[0027] In another optional, and alternative, embodiment a polypeptidesuch as neurotoxin is produced in an inactive form using the Pichiaexpression system described herein. To the best of Applicants knowledge,the prior art fails to teach or suggest the preparation of a toxin ininactive form by the route of cytoplasmic expression in yeast.

[0028] The method and composition of the present invention provide aunique and valuable tool for the synthesis and recovery of bioactivepolypeptides in a manner cap able of diminishing undesirable activity,yet retaining other useful properties of the polypeptide (such asimmunogenicity and antiviral activity).

DETAILED DESCRIPTION

[0029] As used herein, the following words (and inflections thereof) andterms will have the meanings ascribed to them below:

[0030] “bioactive” will refer to a polypeptide capable of eliciting atleast one biological response when administered in vivo.

[0031] “polypeptide” will refer to any biomolecule that is made up, atleast in part, of a chain of amino acid residues linked by peptidebonds.

[0032] “inactive” will refer to a polypeptide that is provided in a formin which at least one form of its bioactive responses is substantiallyterminated or decreased to a desired extent.

[0033] “neurotoxin” will refer to a bioactive polypeptide wherein atleast one activity (e.g., binding to the acetylcholine receptor)produces a toxic effect on the nervous system of a mammalian host.

[0034] The method of the present invention involves an initial step ofidentifying a bioactive polypeptide having a tertiary structure in whichbioactivity is dependent, at least in part, on the formation of one ormore disulfide bridges between cysteine residues. Typically, thepolypeptide will be one that is naturally secreted in the course of itssynthesis, since it is the secretion process that will provide thenecessary posttranslational steps, including disulfide bond formation.Preferably, the polypeptide is one that is stable when recovered andthat retains other desirable properties in the unfolded state, such asimmunogenicity and/or antiviral, anti-tumor or wound healing activity.

[0035] The amino acid sequence and tertiary structure of a number ofbioactive polypeptides is known. Suitable polypeptides include those inwhich one or more disulfide bridges are known to form in the naturalconfiguration, and in which such bridge(s) are necessary for thebioactivity of the polypeptide. Such bridges can be of either anintramolecular (i.e., within a single polypeptide) nature and/or anintermolecular (e.g., between discrete subunits) nature.

[0036] Secreted or cell-surface proteins often form additional covalentintrachain bonds. For example, the formation of disulfide bonds betweenthe two —SH groups of neighboring cysteine residues in a foldedpolypeptide chain often serves to stabilize the three-dimensionalstructure of the extracellular proteins. Protein hormones such asoxytocin, arginine vasopressin, insulin, growth hormone and calcitonin,all contain disulfide bonds. Enzymes such as ribonuclease, lysozyme,chymotrypsin, trypsin, elastase and papain also have their tertiarystructure stabilized by disulfide bonds. Besides the bioactive proteinslisted above, there are numerous other proteins that contain disulfidebonds, such as the immunoglobulins (IgA, IgD, IgE, IgM), fibronectin,MHC (major histocompatible complex) molecules and procollagen. Manypolypetides from animal venoms also contain disulfide bonds.

[0037] In a preferred embodiment, the method of the present invention isused to prepare inactivated forms of neurotoxins, and more preferablyneurotoxins from amongst the four groups provided below. As describedabove, those in Group I typically affect the presynaptic neurojunction,those in Group II typically affect the postsynaptic neurojunction, andthose in Group III typically affect ion channels. Lastly, there are alsoincluded toxins known only to have a toxic affect by causing membranedamage. Neurotoxins Membrane-damaging toxins Group I Group II Group IIInotexin α-conotoxin dendrotoxins myotoxins β-bungarotoxin α-cobrotoxinscorpion toxins cardiotoxins crotoxin erabutoxin μ-conotoxins mellitintaipoxin α-cobratoxin sea anemone toxins phospholipases textilotoxinα-bungarotoxin α-latrotoxin

[0038] The method involves a further step of preparing or isolating acorresponding gene (e.g., a cDNA strand) encoding the polypeptide. Usingthe primary amino acid sequence discussed above, and in view of thepresent teaching, those skilled in the art will appreciate the manner inwhich such polypeptides can be synthesized using genetic engineeringtechniques. Generally, and preferably, one or more of the native control(e.g., leader) sequences of the desired cDNA are removed and replacedwith one or more corresponding sequences in order to facilitate thedesired expression.

[0039] Polypeptide components from animal venoms, for instance, can beobtained from the animals themselves or from other sources, or they canbe created in the laboratory using conventional protein engineeringtechniques. In the former approach, animals are induced by mechanical orelectrical stimuli to release venom from their glands, which travelsthrough a venom canal and out the fang or stinger. The venom iscollected and various constituents of the venom are purified byconventional chromatographic techniques.

[0040] In the latter approach, constituents from the venom aresynthesized by cloning the genes encoding the various polypeptideelements and expressing these genes in heterologous host systems such asbacteria, yeast or higher eucaryotic cell lines. Yeast expressionsystems are presently preferred, since they tend to provide an optimalcombination of such properties as yield and adaptability to human useproducts.

[0041] Expressed products are then purified from any other contaminatinghost polypeptides by means of chromatographic techniques similar tothose used to isolate the polypeptides directly from the venom.

[0042] There are significant advantages to the use of host systems otherthan the venomous animals to obtain the venom components. The danger tohuman lives in obtaining the venom from the animal is eliminated. Therewill no longer be a need for the costly animal husbandry required tomaintain venomous animals for venom extraction. The quantities ofmaterials that can be obtained from the genetic engineering approach canbe one or more orders of magnitude greater than the quantities that canbe derived from the venom itself. Moreover, once the gene(s) is clonedand expressed, it can be used to provide a continual, reproduciblesource in the form of a bacterial, yeast or higher eucaryotic cell lineseed culture.

[0043] Seed cultures can be stored and transported in the frozen state,lyophilized, or, in some cases, plated on media. Also, the use ofgenetic engineering tools will enable those skilled in the art tomanipulate the genes for the purpose of altering the polypeptide productin any fashion feasible. Using the method of the present invention, incombination with available tools for protein engineering (e.g.,site-directed mutagenesis), those skilled will be able to prepare abioactive polypeptide having any desired level of toxicity, whethernon-toxic, or of diminished, equal or greater toxicity than the nativeform.

[0044] The method of the invention provides a further step of expressingthe cDNA under conditions in which the polypeptide is recovered in aninactive form due to the failure to form one or more disulfide bridges.As described in greater detail below, this step involves the avoidanceof posttranslational processes that would otherwise serve to form suchlinkages.

[0045] Optionally, and preferably, the method provides a further step oftreating the inactivated bioactive polypeptides in order to retain thecysteine residues and prevent the spontaneous formation of disulfidebonds. A preferred treatment includes ozone treatment, in the mannerdescribed herein. Ozonation affects the cysteine residues by convertingthe pendent sulfhydryl (—SH) groups to corresponding —SO3X groups,which, unlike the sulfhydryl groups, are unable to form a disulfidebridge. Such treatment is not necessary, however, for those inactivatepolypeptides that are found to not spontaneously reform, and thatprovide the desired activity. Ozonation is preferred for polypeptidessuch as neurotoxins, where Applicant has shown that upon cleavage andozonation of the sulfhydryl groups, native neurotoxins are both stableand active.

[0046] The invention further provides a bioactive polypeptide that hasbeen rendered inactive by virtue of the failure to form one or moredisulfide bridges. Such polypeptides can be stably stored and used underconditions in which disulfide bonds are prevented from spontaneouslyreforming.

[0047] In yet another aspect, the invention provides a method ofadministering a bioactive polypeptide to a host, comprising the step ofproviding the polypeptide in an inactive form and within a suitablecomposition, and administering the composition to a host. In a relatedaspect, the invention provides a host having administered such apolypeptide. Compositions of the present invention can be used for avariety of purposes. Compositions are particularly useful in situationscalling for a polypeptide in a form that is as close to native aspossible, yet without an unwanted bioactivity.

[0048] The invention may be more easily appreciated by reference to thefollowing non-limiting examples in which parts are expressed by weightunless otherwise indicated. The disclosures of each of the referencescited herein are incorporated by reference.

EXAMPLES Example 1 Isolation of Gland Tissue for RNA Extraction

[0049] The following protocol was used to clone the gene encodingα-cobratoxin from the venom of Naja naja siamensis.

[0050] (a) Recovery of Venom

[0051]Naja naja siamensis snakes were obtained from Siam Farms, Bangkok,Thailand. Animals were shipped to and housed at Ventoxin, Inc.,Frederick, Md. USA. The venom glands from N. n. siamensis animals weresurgically removed and used to prepare mRNA for generating a cDNAlibrary. Snakes were placed on a schedule for milking (venomextraction). They were milked on day 1 and eight days later milked asecond time. On the 2nd or 3rd day, they were anesthetized with sodiumpentobarbital and their glands removed (Vandenplas et al., 1985). Glandtissue was quickly cut into small pieces and immediately frozen inliquid nitrogen. Samples were kept at −70° C. until use.

[0052] (b) RNA Isolation

[0053] Total RNA was isolated from gland tissue by using a standardguanidinium/hot phenol method (Feramisco et al., 1982). Frozen glandtissues (5 g) were placed in a polytron mixer and 10 ml of Solution A(guanidinium isothiocyanate mixture) was added to the tissue. Solution Awas prepared by resuspending 100 g of guanidinium isothiocyanate in 100ml of deionized water, 10.6 ml of 1 M Tris-Cl (pH 7.6), and 10.6 ml of0.2 M disodium ethylene diamine tetraacetate (EDTA). It was stirredovernight at room temperature.

[0054] The solution was then warmed while stirring to 60-70° C. for 10min to assist dissolution. Any insoluble material remaining was removedby centrifugation at 3000 g for 10 min at 20° C. To the guanidiniumisothiocyanate solution, was added 21.2 ml of 20% sodium laurylsarkosinate and 2.1 ml of β-mercaptoethanol to the supernatant and thevolume was brought to 212 ml with water. The final solution was filteredthrough a disposable Nalgene filter and stored at 4° C. in a tightlysealed, brown glass bottle.

[0055] The glands were mixed in the polytron mixer at 4° C. until mostof the tissue had been disrupted (about 3-5 min.). The gland solutionwas placed in a 50 ml polypropylene centrifuge tube and 20 ml more ofthe guanidinium isothiocyanate mixture was added. The mixture wasbrought to 60° C. and passed through a syringe fitted with an 18 gaugeneedle. This shearing technique was repeated 2 to 3 times or until theviscosity of the suspension was reduced. An equal volume of ultra pureliquid phenol preheated to 60° C. was added to the tissue suspension andthis was again passed through the syringe 2 to 3 times.

[0056] At this point, 0.5 volume of Solution B (0.1 M sodium acetate (pH5.2), 0.01 M Tris-Cl (pH 7.4), 0.001 M. EDTA) was added to the emulsionand mixed. An equal volume of chloroform/isoamyl alcohol (24/1 v/v) wasadded and the mixture shaken vigorously for 15 min. while maintainingthe temperature at 60° C. The mixture was cooled on ice and centrifugedat 2000 g for 15 min. at 4° C. The aqueous phase, containing the RNA,was recovered and reextracted with phenol/chloroform. To the aqueousphase was added 2 volumes of absolute ethanol and the mixture was storedat −20° C. overnight. All glassware used in extracting and working withRNA had been baked at 250° C. for at least 4 h. Sterile, disposablepolypropylene plasticware is essentially free of RNase and can be usedfor the preparation and storage of RNA without pretreatment.

[0057] The RNA was recovered by centrifugation was dissolved in 30 ml ofSolution C (0.1 M Tris-Cl, pH 7.4, 0.05 M NaCl, 0.01 M EDTA, 0.2% (v/v)sodium dodecyl sulfate (SDS)). Proteinase K was added to a finalconcentration of 200 ug/ml and incubated at 37° C. for 2 h. The solutionwas then heated to 60° C. and 0.5 volume of phenol, preheated to 60° C.,was added and mixed vigorously with the RNA-containing solution.Chloroform (0.5 volume) was added to the solution and again mixedvigorously at 60° C. for 10 min. The solution was cooled on ice for 10min. and then centrifuged at 2000 g for 15 min.

[0058] The aqueous phase was recovered and re-extracted one more timewith phenol/chloroform at 60° C. The aqueous phase was recovered andreextracted twice with chloroform at room temperature. To the aqueousphase was added 2 volumes of absolute ethanol and put at −20° C.overnight. The nucleic acids were precipitated by centrifugation and thepellet rinsed with 70% cold ethanol. RNA was stored at −70° C. in 70%ethanol until used. When the RNA was ready to be used, it wascentrifuged, dried and resuspended in Rnase-free sterile water.

[0059] (c) mRNA Purification

[0060] Poly(A)+ RNA was enriched by passage over an oligo(dT)-cellulosecolumn using a conventional method (Aviv and Leder, 1972). Commercialoligo(dT)-cellulose was equilibrated with sterile, RNase-free Solution D(0.02 M Tris-Cl, pH 7.6, 0.5 M NaCl. 0.001 M EDTA and 0.1% (v/v) SDS). A1.0-ml bed-volume of equilibrated matrix was poured into either anRnase-free disposable polypropylene column or siliconized RNase-freepasteur pipette. The matrix was washed with 3 column volumes of (1)Rnase-free sterile water; (2) 0.1 M NaOH containing 0.005 M EDTA; and(3) sterile water. The column effluent should have a pH less than 8. Thecolumn was then washed with 5 volumes of sterile Solution D.

[0061] The RNA isolated as described above was heated to 65° C. for 5min and a 2× concentration of an equal volume of Solution D was added tothe RNA solution. The sample was cooled to room temperature and loadedonto the oligo(dT)-cellulose column. The flow-through from the columnwas heated to 65° C., cooled to room temperature, and reapplied to thecolumn. The column was washed with 10 volumes of Solution D followed by4 column-volumes of Solution D containing 0.1 M NaCl. The poly(A)+ RNAwas then eluted with 2-3 column volumes of sterile Solution E (0.01 MTris-Cl, pH 7.5, 0.01M EDTA and 0.05% (v/v SDS).

[0062] Typically, NaCl was added to the mRNA to obtain a saltconcentration of 0.5 M, and the mRNA was repurified on a second passageover the oligo(dT)-cellulose column using the same procedures asdescribed for the initial column run. Sodium acetate (NaOAc) (3M, pH5.2) was then added to the mRNA from the second column run to obtain afinal concentration of 0.3 M NaOAc. Cold absolute ethanol (2.5 volumes)was added to the mRNA solution and the solution was placed at −20° C.overnight. The N. n. siamensis mRNA was then centrifuged at 12,000 g,the pellet washed with cold 70% ethanol, and stored in 70% ethanol at−70° C. until used. The yield of mRNA from 5 g of gland tissue was 16μg.

[0063] (d) Construction of a N. n. siamensis cDNA Library.

[0064] Complementary DNA (cDNA) was prepared from 5 μg of N. n siamensismRNA (Guber and Hoffman, 1983) using commercially-available cDNAsynthesis kits. A variety of sources provide cDNA synthesis kits thatare useful for such purposes. In this particular case, cDNA synthesiskit, EcoR I/Not I adaptors, T7 sequencing kit, Deaza T7 sequencingmixes, and restriction enzymes were obtained from Pharmacia (Piscataway,N.J.).

[0065] A lambda ZAP II/EcoR I CIAP treated vector kit and Gigapack IIGold packaging extract were obtained (Stratagene, LaJolla, Calif.), aswas a “GeneAmp PCR reagent kit” (Perkin-Elmer Cetus, Norwalk, Conn.).Oligonucleotides used for screening cDNA libraries and as primers forpolymerase chain reactions (PCR) and dideoxynucleotide sequencing weresynthesized on a Biosearch 8700 DNA synthesizer by 13-cyanoethylphosphoramidite chemistry and purified on Oligo-Pak columns(MilliGen/Biosearch, Burlington, Mass.).

[0066] A protocol for the cDNA synthesis is provided in “You-Prime cDNASynthesis Kit Instructions”, Pharmacia LKB Biotechnology, the disclosureof which is incorporated herein by reference. (See, in particular, pages12, 13, 18, 19 and 29 and Procedures A, B and D thereof for theprototypical procedure.) Using procedure B, hemiphosphorylated adaptorscontaining Not I and EcoR I restriction enzyme sites were ligated to thetermini of the synthesized, double-stranded cDNA prepared in ProcedureA. After purification of the cDNAs (Procedure D), the N. n. siamensiscDNA were inserted into EcoR I-predigested, phosphatased Lambda ZAP IIarms and packaged into viable phage particles by using packagingextracts. The latter was accomplished using a commercially available kitfrom Stratagene (LaJolla, Calif.) (Catalog #236211, “Predigested LambdaZAP II/EcoR I Cloning Kit”).

[0067]N. n. siamensis cDNA was ligated to Lambda ZAP II arms using theprocedure on page 3 of the Strategene instructions (substituting thetest insert for the N.n. siamensis cDNA). The ligated sample was thenpackaged into viable phage particles using a “Gigapack Gold” packagingextract from Strategene (product insert, page 4). The recombinantbacteriophage was used to infect E. coli host strain, XL1-Blue, whichgenerated the primary cDNA library. The primary library containedapproximately 1.35×10⁵ pfu/μg mRNA.

[0068] (e) Isolation of α-cobratoxin CDNA from the cDNA Library andSubcloning of cDNA Inserts from Lambda ZAP II Clones

[0069] Approximately 100,000 plaques from an amplified cDNA library wereanalyzed for sequences encoding α-cobratoxin using a degenerateoligonucleotide probe prepared from the known amino acid sequences ofa-cobratoxin. The probe (LAS 1) was prepared as follows:

[0070] 5′—GGI CAI GTI TGT/C TAT/C ACI AAA/G ACI TGG TGT/C GAI GCI TTITG—3′

[0071] The oligonucleotide probe above was end-labelled on the 5′ endusing [³²P]-γ-ATP and T4 polynucleotide kinase using standard protocols(Sambrook et al. 1989). The library was screened for the presence ofα-cobratoxin cDNA on nitrocellulose filters according to standardprocedures (Sambrook et al., 1989). Filters were prehybridized for 4 hat 42° C. in 6×SSC (90 mM sodium citrate containing 0.9 M NaCl, pH 7.0),containing 1× Denhardt's and 100 mg/ml sonicated and denatured salmonsperm DNA. Filters were then hybridized in 4× SSC, pH 7.0, containing 1×Denhardt solution (50×5 g ficoll, 5 g polyvinylpyrrolidone, 5 g bovineserum albumin/500 ml water) and the radiolabelled oligonucleotide probefor 16 h at 42° C.

[0072] Successive washes were performed in 2×SSC, pH 7.0, at 37° C. for30 min before autoradiography for 16 h at −70° C. using X-AR film withintensifying screens. Double-stranded cDNA inserted into the multiplecloning site (MCS) of pBluescript SK-contained within lambda ZAP II,were removed as phagemids by an in vivo excision process designed byStratagene (LaJolla, Calif.) (see Stratagen insert, page 7, “In VivoExcision Protocol”). Colonies from the in vivo excision were selected byampicillin resistance, propagated, and the phagemids were isolated byalkaline extraction (see pp. 368-369, “Analysis Lysis Method”). The sizeof the inserts from the recombinant phagemids were measured on agarosegel electrophoresis after digestion with the restriction enzyme, EcoR I.

[0073] (f) Characterization of the α-Cobration cDNA by Asymmetric PCRand DNA Sequencing

[0074] The template for asymmetric PCR was double-stranded pBluescriptSK-containing cDNA inserts of approximately 400 bp. Oligonucleotidesdesignated as LAS 2 (5′ GAGTTAGCTCACTCATTAGGC 3′) and LAS 3 (5′ATT-TTCATTCGCCATTCAGGC 3′) were used as primers in asymmetric PCR (see“T7 Sequencing Kit Instructions”, Pharmacia LKB Biotechnology”). Sangerdideoxynucleotide sequencing employed T7 DNA polymerase according to themanufacturer's protocol accompanying the T7 Sequencing (TM) Kit ofPharmacia LKB Biotechnology. N. n siamensis cDNA template, and theprimers (LAS 4 and LAS 5) were as described below. Single stranded DNAwas used as a template. Programs for sequence analysis fromIntelligenetics,Inc. (Mountain View, Calif.), including GENED, SEQ, andIFIND, were used on a VAX from Digital Equipment Corp. (Maynard, Mass.).One of the cDNAs sequences encoded α-cobratoxin (identified as Naja najakaouthia cDNA library clone “NNK III 6.2”). The α-cobratoxin cDNA was anincomplete gene in that the leader sequence coding for the snake signalsequence was incomplete (−1 to −20) lacking an in initiation codon(ATG). For purposes of expression, this was immaterial, since the leadersequence was replaced with a functional start codon and restrictionenzyme site (as described herein with reference to expression of cDNA inyeast).

[0075] The gene encoding α-cobratoxin could also have been preparedusing the genetic coding sequence for the known amino acid sequence ofthe protein, and synthetically constructing a suitable gene usingautomated biochemical techniques.

[0076] The PCR-derived DNA was resuspended in TE buffer (20 mM tris-CL,1 mM EDTA, pH 7.5) and cleaved with the restriction enzyme, EcoR I (seeGibco product insert for EcoR I catalog #15202-013, restriction enzymeassay for EcoR I). The yeast DNA vector (pHILD4) was also taken,resuspended in TE buffer and cleaved with EcoR I.

[0077] The vector DNA was cleaved in the same manner as the PCR-derivedDNA (see Gibco instructions). After digestion with EcoR I, thePCR-derived DNA and yeast vector DNA was purified by the addition of anequal volume of phenol/chloroform (50/50 v/v), vortexing, andcentrifugation in a microfuge (12,000 g). A second chloroform extractionwas performed (equal volume of CHCl₃ and sample), vortexing,centrifugation and ethanol precipitation. Ethanol precipitation wasperformed by adding sodium chloride to the sample (0.2 M finalconcentration) and 2.5 volumes of cold ethanol. After mixing, the samplewas placed on dry ice for 15 min, then centrifuged at 4° C. in amicrofuge (12,000 g) for 15 min. The DNA pellet was dried under vacuum.

[0078] Both of the EcoR I-treated DNAs were resuspended in TE buffer andcovalently joined together using T4 DNA Ligase (see insert materials,Gibco BRL, Cat. # 5224SC, T4 DNA Ligase). The ligated DNA was used totransform competent E. Coli cells (see Enclosure 10 for transformationconditions). Transformants growing on TB agar (Terrific Broth+agar)containing ampicillin (100 μg/ml) were isolated and the recombinant DNAanalyzed by restriction enzyme analysis.

[0079] Optionally, the DNA can be purified from E. coli cells, e.g., inthe manner described in “Wizards Maxipreps DNA Purification System”,Promega. Recombinant DNA from clones harboring the α-cobratoxingene/pHILD4 construct was used for integration into the yeast, Pichiapastoris.

[0080] (g) Cloning and Cytoplasmic Expression

[0081] Expression of the α-cobratoxin gene in the vector, pHILD4 yieldsa cytoplasmic product that lacks posttranslational modifications,including disulfide bond formation.

[0082] Suitable techniques for cloning and expressing genes into Pichiapastoris have been developed by the Phillips Petroleum Company andcompiled in “Pichia Expression Kit—A Manual of Methods for Expression ofRecombinant Proteins in Pichia pastoris”, which was prepared byInvitrogen and accompanies their expression kit having catalog#K1710-01.

[0083] The gene encoding α-cobratoxin from amino acids +1 to +71 can beremoved from the cDNA by using the following polymerase chain reactionprimers:

[0084] (a) 5′ sense primer (LAS 4)=5′-GGATCC GAATTC ACG atg [ATA AGAACA]-3′(36 mer) and

[0085] (b) 3′ antisense primer (LAS 5)=5′-CCTAGG GAATTC TTA TCA [AGG aTGG]-3′(36-mer).

[0086] Recombinant DNA prepared as described herein was treated with SstI restriction enzyme under the same reaction conditions as describedabove with respect to EcoR I, except using reaction buffer No. 2described in the above-captioned Gibco EcoR I product insert. Therestricted DNA is purified by the addition of an equal volume ofphenol/chloroform (50/50 v/v), vortexing, and centrifugation in amicrofuge (12,000 g).

[0087] A second chloroform extraction was performed (equal volume ofCHCl₃ and sample), vortexing, centrifugation and ethanol precipitation.Ethanol precipitation was performed by adding sodium chloride to thesample (0.2 M final concentration) and 2.5 volumes of cold ethanol.After mixing, the sample was placed on dry ice for 15 min, thencentrifuged at 4° C. in a microfuge (12,000 g) for 15 min. The DNApellet was dried under vacuum and resuspended in TE buffer.

[0088] The DNA pellet is then integrated into the chromosome of Pichiapastoris strain GS 115 using conventional procedures for integratinggenes into Pichia pastoris (e.g., p. 29-38, “Growth of Pichia forSpheroplasting”) and expressing the integrated genes (pp. 41-45,“Expression of Recombinant Pichia strains”).

Example 2 Recovery and Yield

[0089] A fermentation of a cytoplasmically-expressing clone harboringthe gene encoding α-cobratoxin can be performed in a 5 L New BrunswickBioFlo III fermentor. The size of the fermentation can be scaled up ordown depending on the requirement for product. For a 5 L batch, a frozenseed culture containing the α-cobratoxin construct is used to inoculate10 ml of MGY media (see attached media recipe) in a test tube. After 18to 20 hours growth at 30° C., 0.5 ml is used to inoculate 50 ml of MGYin a 250 ml flask. After 36 to 38 hours of growth, the entire 50 ml isused to inoculate the 5 L fermentor. The fermentation is performed in abasal salt medium with 26.7 ml 85% phosphoric acid, 0.93 g/L calciumsulfate-2H₂O, 18.2 g/L potassium sulfate, 14.9 g/L magnesium sulfate,4.13 g/L potassium hydroxide, 40 g/L glycerol and 2 m/L of basal salts(PTM) are added. PTM basal salts consist of 2.0 g cupric sulfate, 0.08 gsodium iodide, 3.0 g magnesium sulfate, 0.2 g sodium molybdate, 0.02 gboric acid, 0.5 g cobalt chloride, 7.0 g zinc chloride, 22 g ferroussulfate, 0.2 g biotin and 1 ml sulfuric acid per liter. The fermentationculture is fed with a 50% solution of glycerol in deionized water, whilethe methanol feed solution is 100% methanol with 2 ml of PTM basal saltsand 1 mg biotin per liter. “Structol” brand antifoamer can be used asantifoam control; the pH during the glycerol phase is maintained at pH5.0 using 30% ammonium hydroxide; dissolved oxygen is controlled above25% saturation by supplementing with pure oxygen.

[0090] A standard fermentation procedure is followed which includes aninitial batch phase followed by a 4 hour glycerol fed-batch with a feedrate of 15 ml/L/h of a 50% glycerol solution. At the completion of theglycerol fed-batch phase the methanol induction phase is started. Therate of methanol feeding is increased gradually from 3.5 to 12 ml/L/hwithin 6 to 8 hours and maintained at 12 ml/L/h. Samples are takenduring fermentation for measuring optical density at 600 nm, cell dryweight and SDS-PAGE analysis.

[0091] Yeast cells are recovered from the fermentation bycentrifugation. Cells are washed in breaking buffer (50 mM NaH₂PO₄, 1 mMEDTA, 5% glycerol, 1% PMSF, pH 6.0), and resuspended in the same bufferprior to disruption in an APV Matnon Gaulin 30CD pilot scalehomogenizer. Cell debris is removed by centrifugation and a PEIprecipitation is performed on the cell extract in order to removeendogenous nucleic acids. Polyethyleneimine (PEI) (10%) is added to thecell extract to obtain a final concentration of 0.4% PEI. The mixture isallowed to sit for 3 to 5 hours at 4° C. with stirring. The mixture iscentrifuged at 27,000×g for 15 min and the supernatant is dialyzedagainst 50 mM NaH₂PO₄, pH 6.0 at 4° C. The recombinant product ispurified by ion exchange (e.g., cationic exchange matrix) and molecularsieve chromatography.

[0092] There have been a number of heterologous proteins produced usingthe Pichia pastoris expression system. The levels of expression fromintracellularly expressed proteins has ranged from 0.3 to 12 g/Ldepending on the protein expressed (Biotechnology 11, 905-910 (1993)).The level of expression is usually dependent on such factors as thegenetic construct itself, cell copy number and fermentation optimization(e.g., cell density, optimal pH and dissolved oxygen concentration).Yields from an α-cobratoxin gene expressed intracellularly in Pichiapastoris will typically fall in the range stated above.

Example 3 Ozonation

[0093] Ozone (O₃), a powerful oxidant, is used for water disinfection.In the course of the present invention, ozone treatment is preferablyused to treat the recovered, inactive polypeptide in order to render itincapable of spontaneous reformation. Optionally, ozonated pure watercan be used to itself selectively break the disulfide bonds of a formedpolypeptide in order to provide an inactive, denatured, and stable formthereof.

[0094] Ozone treatment can be used to quickly provide microbialsterilization and disinfection, organic compound destruction, andconversion of iron or manganese salts to insoluble oxides which can beprecipitated from the water. The major reaction byproducts are water,oxygen and carbon dioxide. For environmental and safety concerns,unreacted or residual ozone should be monitored. A number of UVspectrophotometric methods can be used to determine the level of ozonein water or physiological saline. Ozone has an absorption peak at 260 nmwhereas oxygen does not absorb at this wavelength. When ozoneconcentration was measured ice water (1° C.±1° C.) by three differentcolorimetric methods, the absorbance coefficient in ozone at 260 nm asA_(1 cm) ^(1 mg/L) is 0.11.

[0095] A wavelength scan of ozonated water was determined at variousdilutions. Using the same ozonated water, the ozone concentration wasdetermined by Accuvac method described below. Using this, or similarmethods, it is possible to calculate the ozone content of the ozonatedwater in mg of O₃/L.

[0096] A standard curve for the ozonated water was also prepared. Fromthis curve one can derive the absorbance coefficient of ozone in anygiven solution. Table 1 below provides a representative relationshipbetween absorbance coefficients and concentration for ozonated water.

Absorbance coefficient (A)^(mg/l)=(Absorbance at 260 nm)÷(Concentrationof Ozone) TABLE 1 Absorbance of Concentration of Absorbance OzonatedOzone by Coefficient of water at 260 nm Accuvac method mg/L Ozone at 260nm 1.5717 13.48 0.11659 0.628 6.44 0.0975 .39822 2.908 0.1369 .259532.6792 0.0968 .19797 1.722 0.11496 .13605 1.28 0.1062 AVERAGE VALUE 0.11

[0097] Three different calorimetric methods (“Accuvac”, “Alizarin” and“Indigo Trisulphonate” methods) were used for the determination of ozoneconcentration in ice water (1° C. ±1° C.), and compared to absorbance at260 nm. Ozonated water was prepared as described in above. Certain ofthese methods are used by the International Ozone AssociationStandardization Committee.

[0098] Method 1: Alizarin Method

[0099] The method is directly applicable in the range of 0.03 to 0.6mg/L. A stock solution of Alizarin violet 3R is made up as a 0.2 mMsolution. Disperse 124.45 mg of the dye into an aliquot of distilledwater in a 1 liter volumetric flask. Mix magnetically overnight. Add 20mg of analytical grade sodium hexametaphosphate, 48.5 g of analyticalgrade ammonium chloride and 1.6 g of ammonia expressed as NH₃. Dilutewith distilled water to 1 liter and stir overnight. A 10-fold dilutionof this solution has an absorbance of 0.155 cm⁻¹.) 20 ml of the reagentsolution is introduced into each of two 200 ml volumetric flasks. Fillone flask with ozone free water. Fill the other flask with the samplewater by introducing the sample below the surface of the dye solution toprevent ozone loss by degassing. When measured, the difference inabsorbance at 548 nM is 2810 L/M/cm. This equates to the expression:

mg/L O₃=Total volume (200 ml)×(change in absorption)÷(Cell length (1cm)×0.059×volume of sampled water (180 ml))

[0100] Method 2: Indigo Trisulphonate Method

[0101] The method is directly applicable in the range of 0.01 to 0.1mg/L of ozone in water. A stock solution of indigo-trisulphonate is madeup as a 1 mM solution by dispersing the dye into a solution ofanalytical grade phosphoric acid at a concentration of 1×10⁻³ M. A100-fold dilution of this solution has an absorbance of 0.16+/−0.01/cmat 600 nm and should be discarded if the absorbance is lower than 80% ofthe starting value. Normal stability lasts one month. As a dilutedreagent, 20 ml of the stock solution is diluted to 1 liter together with10 g of analytical grade NaH₂PO₄ and 7 ml concentrated analytical gradeH₃PO₄ (stability of the diluted solution: one week).

[0102] In use, 10 ml of diluted reagent solution is introduced into eachof two 100 ml volumetric flasks. Fill one flask with ozone free water(e.g. distilled water). Fill the other flask with the sample water byintroducing the sample below the surface of the dye solution to preventozone loss by degassing. Measure the difference in absorbance at 600 nmbetween blank and sample with 5 or 10 cm cells. The measurement is to bemade as soon as possible but preferably within 4 hours. The pH value ofthe measured solution must be lower than 4.

[0103] The proportionality constant is 0.42+/−0.01/cm/mg/L ozone, whichis equal to a difference in absorbance of 20 L/M/cm (Stoichiometry isconsidered as 1:1). mg/L (O₃)=(total volume (100 Ml)×Change inabsorption)÷(cell length (cm)×0.42×Volume of sampled water (90 ml))

[0104] Method 3: Accuvac Method

[0105] As ozone reacts quantitatively with indigo trisulfonate (Blueindigo dye), the color of the solution fades. Color intensity isinversely proportional to the amount of ozone present, is then measuredat 600 nm with a spectrophotometer. The reagent is formulated to preventinterference from any chlorine residual which may be present. The methodis directly applicable in the range of 0 to 0.25 mg/L.

[0106] In use, gently collect at least 40 ml of sample in a 50 mlbeaker. Collect at least 40 ml of ozone free water (Blank) in anotherbeaker. Fill one Indigo ozone reagent Accuvac ampule with the sample andone ampule with the blank. This is done by immersing the ampule in thebeaker which has the sample. Quickly invert the ampules several times tomix. Take an aliquot of the samples and read at 600 nm inspectrophotometer.

[0107] Read a blank value as X at 600 nm. 0.125 mg/L O₃ should haveabsorbance of x/2

g/L of O₃=(0.125×O.D. of the blank value/2)÷(O.D. of the sample at 600nm×Dilution factor) TABLE 2 OZONE CONCENTRATION METHOD (mg/L of water)NOTE Accuvac 13.676 Alizarin 16.8 Indigo - 15.85 Trisulphonate UVabsorption 15.45 (Abs/A^(1 cm/mg/1)) 1.710/.11 at 260 nm

[0108] Table 2 shows the ozone concentration, as determined by thesevarious methods, for aliquots of the same ozonated water. From theresults in TABLE 2 it can be seen that each method providessubstantially the same concentration of ozone. Since all the fourmethods seem to be comparable to each other, the UV absorption method ispreferred since it is simple and inexpensive to perform.

[0109] Ozone was produced by a high voltage discharge using Tri AtomicOxygen Generator (Model No. 3, Serial No. 34 from modem MedicalTechnology Boca Raton, Fla.) The oxygen was passed through the generatorto produce the ozone. Approximately 0.2% of ozone was produced in theequipment at the rate of bubbling used (about 200 ml/min). However, forquantitation studies a sample was taken with each series of experiments.

[0110] Absorption measurements were made in the Beckman DU 650Spectrometer using cm quartz cuvettes. A standard curve was obtained byserially diluting the ozonated water and measuring the absorbance at 260nm. The standard curve was also obtained by using a colorimetric methodusing commercially available Accuvac ampules (From Hach, P.O. Box 389,Loveland, Colo. 80539)

[0111] Saturated ozone water was prepared in the following manner.Oxygen was bubbled at the rate of 200 ml/min to ice water (1° C.±1° C.).The container with distilled water was kept in an ice bath during theozonation. Ozone, bubbled into water or saline, was determined bymeasuring the absorbance at 260 nm. Using a 50 mL sample, it takes aminimum of 30 minutes to reach an absorbance reading of 2.0, althoughthe time is dependent upon the oxygen input.

[0112] Since water that is saturated with oxygen will not becomesaturated with ozone, the flow rate of input oxygen was ideally kept atequal to or less than 200 mL/min. Once the ozonated water reaches anabsorbance of 1.0 to 2.0, serial dilutions of the ice cold ozonatedwater were made and measurements of the absorbance at 260 nm were made.The ozonated water was also used to measure kinetics, and in particular,decay rate over the time. The serially diluted water was used to measurethe ozone concentration by Accuvac method.

[0113] Water ozonated in this manner can be used to oxidize a formedpolypeptide, in order to cleave the disulfide groups and render thepolypeptide inactive. Alternatively, and preferably, the ozonate watercan be used to stabilize a polypeptide that is prepared in an inactiveform by the genetic engineering method described above. In either case,the oxidized peptide can be compared to the original, active toxin usinga variety of methodologies, including animal models and bioassays.

[0114] In a typical approach, the material to be stabilized (e.g.,lyophilized salt free toxin) is weighed into 150 ml plastic bottles,each containing 600 mg of toxin. Approximately 800 ml of pure deionizedwater is allowed to chill in the freezer until ice crystals begin toform. The beaker of pure water is placed in an ice bath and ozonated bybubbling O₃ from an ozone generator connected to an O₂ source.Measurements of OD are taken at 260 nm using a 1 cm light path until anOD₂₆₀ of 2.0 is achieved.

[0115] Sixty ml of ice cold ozonated pure water is added to each bottlecontaining 600 mg of toxin, resulting in a 1 percent solution (aconcentration of 10 mg/ml). While waiting for the powder to dissolve,the bottles are stored in the freezer and ice crystals are again allowedto form. Once in solution, the bottles are placed in an ice bath whereeach bottle is ozonated for 30 seconds by bubbling ozone into thesolution. Ten bottles are done at one time, such that each bottle isozonated for 30 seconds every five minutes. This is done to maintain aneffective level of O₃ and is continued for seven hours.

[0116] Periodic testing is done by injecting mice with the toxinsuspension and monitoring the time to death. When the mice no longer die(after seven hours ozonation) all disulfide bonds have been broken, andthe material has been effectively converted from an active toxin to anatoxic toxoid. It has been noted that if the original ozonated proteinsolution is maintained at 4° C. for 24 hours and, no further ozonationis carried out, the disulfide bonds are likely not going to be broken,and the solution will remain toxic and able to kill mice. Also, whenbacterial or viruses suspensions are added to ozonated water as preparedabove, there is immediate 6-8 log kill. Since bacterial and viral killappears to occur well before oxidation of proteins, ozonated waterprepared in this manner can be used to treat protein-containingformulations (e.g., monoclonal antibody preparations) in order toinactive any remaining animal viruses without damaging the antibodyitself by breaking critical disulfide bonds.

[0117] The oxidized (or stabilized) toxin polypeptide can be compared tothe native alpha neurotoxin in a number of respects. It is found thatthe former is atoxic is mice, while the latter retains full toxicity.The molecular weights as measured on SDS gels are 7380 daltons for boththe primary neurotoxin and the resultant oxidized peptide. Theisoelectric point as measured by iso-electric focusing gels variessubstantially because of the breaking (or stabilized failure to form) ofthe five disulfide bonds creating a net charge change of ten. Theisoelectric point is the pH at which a protein migrates to in anampholyte solution (continuous pH gradient) to which a current isapplied. The primary alpha neurotoxin and resultant oxidized peptidealso show separate peaks when measured by HPLC and FPLC.

[0118] While a preferred embodiment of the present invention has beendescribed, it should be understood that various changes, adaptations andmodifications may be made therein without departing from the spirit ofthe invention and the scope of the appended claims.

What is claimed is:
 1. A method of preparing a composition comprising an inactivated bioactive polypeptide, the method comprising the steps of: a) identifying a polypeptide having a biological activity dependent on the presence of one or more disulfide bridges in its tertiary structure, b) preparing a cDNA strand encoding the polypeptide, c) expressing the cDNA under conditions in which the polypeptide is recovered in an inactive form due to the failure to form one or more disulfide bridges, and d) recovering the inactive polypeptide and formulating it into an aqueous composition.
 2. A method according to claim 1, wherein the composition is suitable for parenteral administration to a host.
 3. A method according to claim 1 wherein the bioactive polypeptide is selected from the group consisting of toxins affecting the presynaptic neurojunction, toxins affecting the postsynaptic neurojunction, toxins those affecting ion channels, and toxins that damage the cell membrane.
 4. A method according to claim 3 wherein the toxins affecting the presynaptic neurojunction toxins are selected from the group consisting of notexin, β-bungarotoxin, crotoxin, taipoxin, textilotoxin and α-latrotoxin.
 5. A method according to claim 3 wherein the toxins affecting the postsynaptic neurojunction are selected from the group consisting of α-conotoxins, α-cobrotoxin, erabutoxin, α-cobratoxin and α-bungarotoxin.
 6. A method according to claim 3 wherein the toxins affecting ion channels are selected from the group consisting of dendrotoxins, scorpion toxins, μ-conotoxins, and sea anemone toxins.
 7. A method according to claim 3 wherein the toxins that damage the cell membrane are membrane-damaging toxin selected from the group consisting of myotoxins, cariotoxins, mellitin, and phospholipases.
 8. A method according to claim 1 wherein the toxin is produced by expressing cDNA under conditions that allow the polypeptide to be recovered with one or more disulfide bridges unformed.
 9. A method according to claim 8 wherein the cDNA is expressed in an expression system selected from the group consisting of bacteria, yeast or higher eucaryotic cell lines.
 10. A method according to claim 9 wherein the expression system is a yeast expression system.
 11. A method according to claim 10 wherein the yeast expression system is selected from the group consisting of Saccharomyces cerevisiae and Pichia pastoris expression systems.
 12. A method according to claim 11 wherein the expression system is a Pichia expression system and the polypeptide is cytoplasmically produced in a manner that allows the product to be recovered in inactive form, without the formation of disulfide bridges.
 13. A method according to claim 12 comprising the further step of treating the recovered polypeptide with ozonated water in order to stabilize the cysteine residues and prevent the formation of disulfide bridges.
 14. A method according to claim 9 wherein the polypeptide is recovered in active form subjected to ozone treatment in order to break the disulfide bridges and render the polypeptide inactive and atoxic.
 15. A composition comprising an atoxic polypeptide prepared according to the method of claim
 1. 16. A composition according to claim 15 wherein the composition is provided in sterile form suitable for parenteral administration to a host.
 17. A method for preparing a bioactive polypeptide in an inactivated form, the method comprising the step of treating the polypeptide with ozonated water under conditions suitable to both oxidize any disulfide bonds in order to form corresponding pairs of cysteine residues, and to then stabilize the resultant cysteine residues and prevent the reformation of disulfide bonds.
 18. An inactivated bioactive polypeptide prepared by a method that comprises the step of treating a bioactive polypeptide with ozonated water under conditions suitable to both oxidize any disulfide bonds in order to form corresponding pairs of cysteine residues, and to then stabilize the resultant cysteine residues and prevent the reformation of disulfide bonds. 